RNA-seq of Homo spaiens: 46,XX LCL
Cells were collected and frozen in Qiagen RNA Protect Cell Reagent until RNA extraction with the Qiagen RNeasy Plus Mini Kit which included a genomic DNA removal step with a gDNA Eliminator Column. 2ul of a 1 to 100 dilution of ERCC Spike-Ins were added per 1ug of RNA.  Libraries were prepared using the Illumina TruSeq mRNA library kit according to manufacturers instructions with the following modifications -  Libraries were size-selected using 2 percent agarose gels on the Sage Science PippinHT system with a capture window of 300-600 bases. The size-selected material was then subjected to one additional cycle of PCR with fresh reagents and P5 P7 primers to ensure all library fragments were fully double-stranded. This step significantly reduced the percentage of smaller fragments co-purifying in the final gel size-selection. After PCR libraries were size-selected a second time on the PippinHT with the same settings.